Psychological and physical stress can alter the immune system in both humans and animals. We have reported that mice subjected to chronic 12-h daily physical restraint for 2 days showed dramatic apoptosis in splenocytes. To identify genes that contribute to the splenocyte apoptosis, we compare gene expression in the spleens of restrained and unstressed mice using oligo microarrays consisting of 226 genes. We report here that mice subjected to chronic 12-h daily physical restraint for 2 days exhibited significantly altered expression of 50 of 226 genes. These genes included pro-apoptotic genes. We selected 5 genes of interest and confirmed the microarray results by real-time PCR. In this study, we identify a potentially important component of pro-apoptotic activity in restraint stress and suggest a possible target for anti-apoptotic therapy to protect splenocytes against stress-induced apoptosis.

No description available.

O córtex pré-frontal medial ventral (CPFMv) é uma estrutura límbica que está envolvida em respostas autonômicas associadas a reações aversivas. O CPFMv é dividido em córtex pré- límbico (PL), córtex infralímbico (IL) e córtex dorsopeduncular (DP). A estimulação elétrica ou química destas regiões causa respostas defensivas e alterações autonômicas tais como respostas cardiovasculares, dependendo da sub-região estimulada. O estresse de restrição (ER) causa alterações hormonais e respostas autonômicas, tais como aumento de pressão arterial (PA) e frequência cardíaca (FC). A ativação de neurônios presentes no CPFMv durante essa situação aversiva, assim como os resultados da inibição farmacológica das sinapses presentes no PL e IL sugerem o envolvimento destas estruturas na modulação das respostas cardiovasculares causadas pelo ER. Entretanto, os possíveis neurotransmissores presentes no vCPFM, envolvidos nesta modulação, ainda não foram elucidados. O sistema opioidérgico central modula o sistema cardiovascular inclusive durante situações aversivas, sendo que tanto receptores quanto peptídeos opióides estão presentes no CPFMv. Considerando o exposto acima, a hipótese a ser testada no presente trabalho foi que a neurotransmissão opioidérgica do PL e IL está envolvida na modulação das respostas cardiovasculares de aumento da PA e FC desencadeadas pelo ER. Assim, a administração de naloxona (antagonista não-seletivo de receptores opióides) no PL ou IL reduziu a resposta pressora e taquicardíaca induzida pelo ER, sendo o perfil da curva dose-inibição em forma de U-invertido. A administração de CTAP (antagonista dos receptores opióides µ) ou nor-BNI (antagonista dos receptores opióides ?) no PL também reduziu a resposta pressora e taquicardíaca induzida pelo ER, de forma semelhante à naloxona, sugerindo o envolvimento desses receptores na modulação das respostas cardiovasculares desencadeadas pelo ER, enquanto que no IL, apenas a administração de nor-BNI reduziu a resposta cardiovascular induzida pelo ER. O tratamento com naltrindole (antagonista ?-seletivo) em ambas as estruturas não alterou a resposta pressora e taquicardíaca gerada pelo ER. A administração de UPF-101 (antagonista ORL-1) no PL potencializou a resposta taquicardíaca, sem alterar a resposta pressora enquanto a administração no IL não gerou efeito. Em resumo, os resultados indicam que o sistema opioidérgico, presente no PL e IL, desempenha papel facilitatório sobre as respostas cardiovasculares induzidas pelo ER, enquanto o sistema nociceptina/orfanina FQ apresentaria papel inibitório. / The ventral medial prefrontal cortex (vMPFC) is a limbic structure involved in the mediation of autonomic responses associated to aversive situations. The vMPFC is divided into prelimbic cortex (PL), infralimbic cortex (IL) and dorsal peduncular cortex (DP). The electrical or chemical stimulation of these regions cause defensive responses and autonomic changes, such as cardiovascular responses, depending on the subregion stimulated. The restraint stress (RS) evokes hormonal and autonomic responses, as well as arterial pressure and heart rate increases. Neuronal activation in the vMPFM was reported during this aversive situation, and the pharmacological inhibition of synapses in the PL and IL has suggested the involvement of these structures in the modulation of cardiovascular responses caused by RS. However, the possible neurotransmitters present in vCPFM that are involved in this modulation have not yet been identified. Opioid peptides and their receptors are present in the CPFMv. Furthermore, the central opioid system is known to modulate the cardiovascular system, even during aversive situations. Therefore, the hypothesis of this study was that PL and IL opioid neurotransmission is involved in the modulation of cardiovascular responses caused by RS. Naloxone (opioid nonselective antagonist) administration in PL or IL reduced the pressure and tachycardiac response evoked by RS, with the dose-inhibition curve having an U-inverset shape. Similar to naloxone, the selective µ-opioid antagonist CTAP and the selective ?-opioid antagonist nor-BNI when administered into the PL also reduced the pressor and tachycardiac response induced by RS, thus suggesting an involvement of these receptors in the modulation of cardiovascular responses evoked by RS, while in the IL, only administration of nor- BNI reduced the cardiovascular response induced by RS. In both structures, the treatment with the selective ?-opioid antagonist naltrindole did not affect the pressor and tachycardic response caused by RS. The pretreatment of the PL with the selective ORL-1 antagonist UPF-101 increased the tachycardic response, without affecting the RSevoked pressor, while the administration of UPF-101 into the IL did not affect the RS-evoked cardiovascular response. In summary, the opioid system in PL and IL appear to play a facilitatory role on the cardiovascular responses induced by RS, while the system nociceptin / orphanin FQ would have an inhibitory role on these responses.

Multiple sclerosis (MS) is an immune-mediated prevalent chronic demyelinating and neurodegenerative disease of the central nervous system that begins with an abrupt onset during early adulthood. MS is idiopathic, but many factors are thought to influence the pathogenesis of the disease, which include genetic, gender and environmental factors. To date, there is much evidence that suggest that both the onset and progression of MS is facilitated by both viral infections and stress. Theiler’s murine encephalomyelitis virus (TMEV) is a picornavirus that upon inoculation into susceptible strains of mice (i.e. SJL and CBA) causes a persistent infection which, in turn, results in an early acute encephalomyelitis followed by a late chronic immune-mediated demyelinating and neurodegenerative disease that pathologically resembles MS. In contrast, resistant mice (i.e C57BL/6 and BALB/c) are able to clear the virus from the CNS, and consequently do not develop chronic demyelination. Previous studies indicated that stress during early infection of susceptible mice can increase CNS viral titers and alter dissemination of TMEV, decrease early cytokine and chemokine expression in the spleen and CNS, and result in an exacerbated late demyelinating disease. The studies herein, focused on the hypothesis that chronic stress during early infection with TMEV infection would lead to drastic immunosuppression of both innate and adaptive arms of immunity, and that this immunosuppression may overcome the genetically controlled resistance of C57BL/6 mice to Theiler’s virus-induced demyelination. In these series of studies, we were able to show that stress, regardless of mouse strain susceptibility, decreases NK cell activity, and increased viral titers at day 1 p.i. Furthermore, after seven days of stress, susceptible mice demonstrated decreased virus specific T-cell effector function in both the CNS and spleens as indicated by a globalized reduction in type 1 and type 2 cytokines, as well as transcription factors. Importantly, these decreased responses were, in part, attributable to the actions of glucocorticoids. However, stress during early infection of C57BL/6 mice did not alter resistance to demyelination. These results begin to shed light on how stress, infection, and genetics can influence the onset of human MS.

Multiple sclerosis (MS) is an immune-mediated prevalent chronic demyelinating and neurodegenerative disease of the central nervous system that begins with an abrupt onset during early adulthood. MS is idiopathic, but many factors are thought to influence the pathogenesis of the disease, which include genetic, gender and environmental factors. To date, there is much evidence that suggest that both the onset and progression of MS is facilitated by both viral infections and stress. Theiler’s murine encephalomyelitis virus (TMEV) is a picornavirus that upon inoculation into susceptible strains of mice (i.e. SJL and CBA) causes a persistent infection which, in turn, results in an early acute encephalomyelitis followed by a late chronic immune-mediated demyelinating and neurodegenerative disease that pathologically resembles MS. In contrast, resistant mice (i.e C57BL/6 and BALB/c) are able to clear the virus from the CNS, and consequently do not develop chronic demyelination. Previous studies indicated that stress during early infection of susceptible mice can increase CNS viral titers and alter dissemination of TMEV, decrease early cytokine and chemokine expression in the spleen and CNS, and result in an exacerbated late demyelinating disease. The studies herein, focused on the hypothesis that chronic stress during early infection with TMEV infection would lead to drastic immunosuppression of both innate and adaptive arms of immunity, and that this immunosuppression may overcome the genetically controlled resistance of C57BL/6 mice to Theiler’s virus-induced demyelination. In these series of studies, we were able to show that stress, regardless of mouse strain susceptibility, decreases NK cell activity, and increased viral titers at day 1 p.i. Furthermore, after seven days of stress, susceptible mice demonstrated decreased virus specific T-cell effector function in both the CNS and spleens as indicated by a globalized reduction in type 1 and type 2 cytokines, as well as transcription factors. Importantly, these decreased responses were, in part, attributable to the actions of glucocorticoids. However, stress during early infection of C57BL/6 mice did not alter resistance to demyelination. These results begin to shed light on how stress, infection, and genetics can influence the onset of human MS.

O córtex pré-frontal medial ventral (CPFMv) é uma estrutura límbica que está envolvida em respostas autonômicas associadas a reações aversivas. O CPFMv é dividido em córtex pré- límbico (PL), córtex infralímbico (IL) e córtex dorsopeduncular (DP). A estimulação elétrica ou química destas regiões causa respostas defensivas e alterações autonômicas tais como respostas cardiovasculares, dependendo da sub-região estimulada. O estresse de restrição (ER) causa alterações hormonais e respostas autonômicas, tais como aumento de pressão arterial (PA) e frequência cardíaca (FC). A ativação de neurônios presentes no CPFMv durante essa situação aversiva, assim como os resultados da inibição farmacológica das sinapses presentes no PL e IL sugerem o envolvimento destas estruturas na modulação das respostas cardiovasculares causadas pelo ER. Entretanto, os possíveis neurotransmissores presentes no vCPFM, envolvidos nesta modulação, ainda não foram elucidados. O sistema opioidérgico central modula o sistema cardiovascular inclusive durante situações aversivas, sendo que tanto receptores quanto peptídeos opióides estão presentes no CPFMv. Considerando o exposto acima, a hipótese a ser testada no presente trabalho foi que a neurotransmissão opioidérgica do PL e IL está envolvida na modulação das respostas cardiovasculares de aumento da PA e FC desencadeadas pelo ER. Assim, a administração de naloxona (antagonista não-seletivo de receptores opióides) no PL ou IL reduziu a resposta pressora e taquicardíaca induzida pelo ER, sendo o perfil da curva dose-inibição em forma de U-invertido. A administração de CTAP (antagonista dos receptores opióides µ) ou nor-BNI (antagonista dos receptores opióides ?) no PL também reduziu a resposta pressora e taquicardíaca induzida pelo ER, de forma semelhante à naloxona, sugerindo o envolvimento desses receptores na modulação das respostas cardiovasculares desencadeadas pelo ER, enquanto que no IL, apenas a administração de nor-BNI reduziu a resposta cardiovascular induzida pelo ER. O tratamento com naltrindole (antagonista ?-seletivo) em ambas as estruturas não alterou a resposta pressora e taquicardíaca gerada pelo ER. A administração de UPF-101 (antagonista ORL-1) no PL potencializou a resposta taquicardíaca, sem alterar a resposta pressora enquanto a administração no IL não gerou efeito. Em resumo, os resultados indicam que o sistema opioidérgico, presente no PL e IL, desempenha papel facilitatório sobre as respostas cardiovasculares induzidas pelo ER, enquanto o sistema nociceptina/orfanina FQ apresentaria papel inibitório. / The ventral medial prefrontal cortex (vMPFC) is a limbic structure involved in the mediation of autonomic responses associated to aversive situations. The vMPFC is divided into prelimbic cortex (PL), infralimbic cortex (IL) and dorsal peduncular cortex (DP). The electrical or chemical stimulation of these regions cause defensive responses and autonomic changes, such as cardiovascular responses, depending on the subregion stimulated. The restraint stress (RS) evokes hormonal and autonomic responses, as well as arterial pressure and heart rate increases. Neuronal activation in the vMPFM was reported during this aversive situation, and the pharmacological inhibition of synapses in the PL and IL has suggested the involvement of these structures in the modulation of cardiovascular responses caused by RS. However, the possible neurotransmitters present in vCPFM that are involved in this modulation have not yet been identified. Opioid peptides and their receptors are present in the CPFMv. Furthermore, the central opioid system is known to modulate the cardiovascular system, even during aversive situations. Therefore, the hypothesis of this study was that PL and IL opioid neurotransmission is involved in the modulation of cardiovascular responses caused by RS. Naloxone (opioid nonselective antagonist) administration in PL or IL reduced the pressure and tachycardiac response evoked by RS, with the dose-inhibition curve having an U-inverset shape. Similar to naloxone, the selective µ-opioid antagonist CTAP and the selective ?-opioid antagonist nor-BNI when administered into the PL also reduced the pressor and tachycardiac response induced by RS, thus suggesting an involvement of these receptors in the modulation of cardiovascular responses evoked by RS, while in the IL, only administration of nor- BNI reduced the cardiovascular response induced by RS. In both structures, the treatment with the selective ?-opioid antagonist naltrindole did not affect the pressor and tachycardic response caused by RS. The pretreatment of the PL with the selective ORL-1 antagonist UPF-101 increased the tachycardic response, without affecting the RSevoked pressor, while the administration of UPF-101 into the IL did not affect the RS-evoked cardiovascular response. In summary, the opioid system in PL and IL appear to play a facilitatory role on the cardiovascular responses induced by RS, while the system nociceptin / orphanin FQ would have an inhibitory role on these responses.

Agmatine is an endogenous amine derived from decarboxylation of arginine catalysed by arginine decarboxylase. Agmatine is considered a novel neuromodulator and possesses neuroprotective properties in the central nervous system. The present study examined whether agmatine has neuroprotective effects against repeated restraint stress-induced morphological changes in rat medial prefrontal cortex and hippocampus. Sprague-Dawley rats were subjected to 6 h of restraint stress daily for 21 days. Immunohistochemical staining with β-tubulin III showed that repeated restraint stress caused marked morphological alterations in the medial prefrontal cortex and hippocampus. Stress-induced alterations were prevented by simultaneous treatment with agmatine (50 mg/kg/day, i.p.). Interestingly, endogenous agmatine levels, as measured by high-performance liquid chromatography, in the prefrontal cortex and hippocampus as well as in the striatum and hypothalamus of repeated restraint rats were significantly reduced as compared with the controls. Reduced endogenous agmatine levels in repeated restraint animals were accompanied by a significant increase of arginine decarboxylase protein levels in the same regions. Moreover, administration of exogenous agmatine to restrained rats abolished increases of arginine decarboxylase protein levels. Taken together, these results demonstrate that exogenously administered agmatine has neuroprotective effects against repeated restraint-induced structural changes in the medial prefrontal cortex and hippocampus. These findings indicate that stress-induced reductions in endogenous agmatine levels in the rat brain may play a permissive role in neuronal pathology induced by repeated restraint stress.

Objective: Stress, either physical or psychological, can modulate immune function. However, the mechanisms associated with stress-induced immune suppression remain to be elucidated. β-Arrestin 2 serves as adaptor, scaffold, and/or signal transducer. The role of β-arrestin 2 in stress-induced immune suppression is not known yet. Methods/Results: Here, we demonstrate that β-arrestin 2 deficiency in mice increases the sensitivity to the chronic stress-induced reduction in the number of splenocytes. Interestingly, the stress-induced suppression of T helper-type (Th) 1 cytokines and the increased production of Th2 cytokines were greatly enhanced in β-arrestin 2-deficient mice compared with wild-type mice. Moreover, inhibition of PI3K in β-arrestin 2-deficient mice exerts an additive effect on the stress-induced reduction in the number of splenocytes. Conclusion: Our study demonstrates that a deficiency in β-arrestin 2 augments stress-induced immune suppression.

Restraint stress, either physical or psychological, can modulate immune function. However, the mechanisms associated with stress-induced lymphocyte reduction remains to be elucidated. We have previously shown that chronic stress induces Fas-mediated lymphocyte reduction. Here, we investigated the mechanisms by which restraint stress modulates lymphocyte reduction. Our data have shown that inhibition of p53 by the p53 inhibitor PFT-α attenuates stress-induced reduction in lymphocyte numbers. These results were verified using p53 knockout mice, suggesting a pivotal role of p53 in this process. In addition our data have indicated that PI3K/nuclear factor kappa B (NF-κB) signaling pathway plays an important role in the stress-induced lymphocyte reduction. Our study thus demonstrates that restraint stress promotes lymphocyte reduction through p53 and PI3K/NF-κB pathways.

Repeated stress or stressful events has been reported to induce anxiety and depression. Differential responses to repeated stress and antidepressant treatments have been observed in males and females. Clomipramine is a tricyclic antidepressant that inhibits the reuptake of serotonin and norepinephrine by indirect actions on the dopaminergic system and limbic-hypothalamic-pituitary-adrenal (LHHA) axis. Its chronic use increases the body's ability to cope with stress; however, high doses can potentiate its side effects on memory, learning, and sensory motor function. Thus, this study investigated sexrelated differential response to repeated restraint stress and chronic administration of clomipramine on parameters of oxidative stress in cerebral cortex, hippocampus and striatum (Manuscript 1) and behavior in rats (Manuscript 1). Male and female rats were divided into control and repeated restraint stress, and subdivided into treated or not with clomipramine during 40 days of stress and 27 days of clomipramine treatment (30mg/kg). The model of repeated restraint stress used in this study increased oxidative damage particularly in striatum and hippocampus of females in higher sensitivity to stress compared to males (Manuscript 1). In this context, the levels of non-enzymatic antioxidant such as the levels of non-protein thiol (NP-SH), and enzymatic antioxidants such as the enzyme superoxide dismutase (SOD), catalase (CAT) and Na+/ K+-ATPase (Manuscript 1) also changed in both sexes. Antidepressant treatment with clomipramine increased the oxidative damage caused by the imbalance between oxidants/antioxidants, particularly in males. Regarding the behavioral activities (Manuscript 1), females subjected to repeated restraint stress had higher anxiety and lower motivation when compared to males. On the other hand, males had impaired non-spatial memory formation through variables such as the short and long term memory as well as a reduction in exploratory and locomotor activity compared to females. Sex differences were also checked as to which antidepressant treatment that potentiate the deficits on the non-spatial memory formation, locomotor and exploratory activities and motivation in males, but in contrast was observed an increase in anxiety and a reduction on the consumption of sweet food in females. Finally, the results of this study indicate that females are particularly sensitive to stress on anxiety and motivation while males on the spatial memory formation as well as locomotor and exploratory acitvities. Antidepressant treatment with clomipramine appears to have neuroprotective effect in females, but cytotoxic in males in what might be observed by the increase in the neurochemical and behavioral deficits. / O estresse pode ser definido como uma variedade de estímulos fisiológicos e psicológicos que podem ter efeitos diretos ou indiretos sobre a função corporal. Neste contexto o estresse repetido ou situações estressantes podem induzir ansiedade e depressão. Diferentes respostas quanto ao estresse repetido e tratamento antidepressivo são observadas em machos e fêmeas. A clomipramina é um antidepressivo tricíclico que inibe a recaptação de serotonina e norepinefrina com ações indiretas sobre o sistema dopaminérgico e eixo límbico-hipotálamo-hipófise-adrenal (LHHA). Seu uso crônico aumenta a habilidade do corpo em combater o estresse, contudo doses elevadas podem potencializar os efeitos colaterais sobre memória, aprendizado e função sensório-motora. Desta forma, este estudo investigou as diferenças sexuais quanto ao estresse repetido por contenção e tratamento crônico com clomipramina sobre parâmetros de estresse oxidativo em córtex cerebral, estriado e hipocampo (Artigo 1 e Manuscrito 1) e comportamento de ratos (Artigo 2 e Manuscrito 1). Machos e fêmeas foram divididos em controle e estresse e subdivididos em tratados ou não com clomipramina durante 40 dias de estresse e 27 dias consecutivos de tratamento com clomipramina (30mg/kg). O modelo de estresse repetido por contenção utilizado neste estudo aumentou o dano oxidativo particularmente em estriado e hipocampo de fêmeas determinando maior sensibilidade ao estresse em comparação aos machos (Manuscrito 1). Neste contexto os níveis antioxidantes não-enzimáticos como tióis nãoprotéicos (NP-SH), enzimáticos tais como a enzima superóxido dismutase (SOD) e catalase (CAT) e Na+/ K+-ATPase (Manuscrito 1) também sofreram alterações em ambos os sexos. O tratamento antidepressivo com clomipramina potencializou o dano oxidativo causado pelo desequilíbrio entre oxidantes/antioxidantes, particularmente em machos. Em relação às atividades comportamentais (Manuscrito 1) fêmeas submetidas à estresse repetido por contenção apresentaram maior ansiedade e menor motivação quando comparado aos machos. Por outro lado, machos apresentaram prejuízo na formação de memória não espacial através das variáveis de memória em curto e longo prazo assim como uma redução na atividade locomotora e exploratória em relação ás fêmeas. Diferenças sexuais foram verificadas também quanto ao tratamento antidepressivo o qual potencializou os déficits sobre a formação da memória não espacial e atividade locomotora e exploratória assim como motivação em machos, mas em contrapartida foi observado um aumento da ansiedade e uma redução quanto ao consumo de alimento doce em fêmeas. Por fim, os resultados deste estudo indicam que fêmeas são sensíveis ao estresse particularmente sobre ansiedade e motivação enquanto machos são sensíveis à formação de memória não espacial assim como atividade locomotora e exploratória. O tratamento antidepressivo com clomipramina na dose utilizada parece ter efeito neuroprotetor em fêmeas, mas citotóxico em machos o que pôde ser observado pelo aumento nos déficits neuroquímicos e comportamentais.